Antioxidative and Antimycotoxigenic Efficacies of Thunbergia laurifolia Lindl. for Addressing Aflatoxicosis in Cherry Valley Ducks

This study aimed to assess the effectiveness of aflatoxin B1 (AFB1) and Thunbergia laurifolia extract (TLE) in the diets of Cherry Valley ducklings. Our investigation covered growth indicators, blood biochemical indices, meat quality, intestinal morphology, immune response, and CP450 enzyme-related gene expression. We conducted the study with 180 seven-day-old Cherry Valley ducks, randomly divided into five dietary treatments. These treatments included a basal diet without AFB1 (T1 group), TLE, or a commercial binder; the basal diet containing 0.1 mg AFB1/kg (T2 group), 0.1 mg AFB1/kg and 100 mg TLE/kg (T3 group), 0.1 mg AFB1/kg and 200 mg TLE/kg (T4 group), and 0.1 mg AFB1/kg and 0.5 g/kg of a commercial binder (T5 group), respectively. Ducklings fed with the T2 diet exhibited lower final body weight (BW), average body weight gain (ADG), and poor feed conversion ratio (FCR) during the 42-day trials. However, all ducklings in the T3, T4, and T5 groups showed significant improvements in final BW, ADG, and FCR compared to the T2 group. Increased alanine transaminase (ALT) concentration and increased expression of CYP1A1 and CYP1A2 indicated hepatotoxicity in ducklings fed the T2 diet. In contrast, ducklings fed T3, T4, and T5 diets all showed a decrease in the expression of CYP1A1 and CYP1A2, but only the T4 treatment group showed improvement in ALT concentration. AFB1 toxicity considerably raised the crypt depth (CD) in both the duodenum and jejunum of the T2 group, while the administration of 200 mg TLE/kg (T4) or a commercial binder (T5) effectively reduced this toxicity. Additionally, the villus width of the jejunum in the T2 treatment group decreased significantly, while all T3, T4, and T5 groups showed improvement in this regard. In summary, T. laurifolia extract can detoxify aflatoxicosis, leading to growth reduction and hepatic toxicosis in Cherry Valley ducklings.


Introduction
Aflatoxins are secondary fungal metabolites, or mycotoxins, primarily produced by toxigenic strains of the fungi Aspergillus flavus and Aspergillus parasiticus [1].These mycotoxins are classified as carcinogenic furanocoumarins and consist of twenty related polycyclic structures [2].Aflatoxin B 1 (AFB 1 ), the most toxic and prevalent aflatoxin, causes oxidative stress, leading to severe hepatoxicity.It also inhibits growth and reproductive performance in poultry, resulting in significant negative effects on animal health, food Toxins 2024, 16, 334 2 of 16 security, and economic trade [3][4][5].Aflatoxins pose a particular problem in hot and dry climates that favor mycotoxigenic fungal growth.Therefore, one of the most severely contaminated areas of AFB 1 in the world is Southeast Asia, especially Thailand, which often experiences higher levels of contamination [6].Previous research indicated that 38.9% of 3206 samples were highly contaminated with aflatoxin, and the prevalence of aflatoxin reached 44.3% in local corn samples [7].
Poultry aflatoxicosis, traced back to the 1960 outbreak of turkey X diseases in the UK, remains a significant threat to the global poultry industry today [8].Aflatoxin-contaminated feeds, exacerbated by climate change, continue to cause poor growth performance, compromised reproductive ability, liver necrosis, and bile duct hyperplasia in poultry, leading to substantial economic losses [9].The detrimental impact extends to bone metabolism, resulting in a weakened skeletal structure and decreased meat yield [10].Among poultry species, ducklings exhibit the highest sensitivity to AFB 1 [11,12] because waterfowls have high levels of unsaturated fatty acids in their body tissues, making them more susceptible to lipid peroxidation induced by AFB 1 [13][14][15].For ducklings, the mortality rates reached 100% at 1 mg/kg AFB 1 [10].Public health concerns arise from aflatoxin residues in poultry products (e.g., eggs and meat), posing risks ranging from mild liver issues to carcinogenesis in consumers [16].These challenges underscore the urgent need for stringent regulations and effective mitigation strategies to safeguard poultry welfare and human health while preserving the economic viability of the poultry industry.
The physical characteristics of aflatoxins include high heat stability and polarity [17].Hence, the efficacy of detoxifying AFB 1 via thermal inactivation is relatively limited.On the other hand, because of the high polarity of aflatoxins, binders exhibit high adsorption ability, making binder supplements the main detoxifying strategy of AFBs in current farms and feed mills [18].However, binders not only remove AFB 1 but also absorb some nutrition compounds (e.g., zinc and vitamin B group) in feed [19].The long-term addition of high amounts of adsorbents can cause zinc deficiency, leading to poultry being unable to stand [20].Phytobiotic feed additives with antioxidant functions appear to be a good choice for detoxifying AFB 1 in poultry.When the feed contains high levels of AFB 1 , phytobiotic feed additives with antioxidant functions can neutralize the mycotoxin toxicities for poultry.When the content is low, they can have multiple uses (e.g., improving immunity and growth traits) for birds [21,22].
Thunbergia laurifolia (Rang chuet) extract (TLE) is widely used for neutralizing toxicities from various toxins [23,24].It is also a common antidote for several poisonous agents in Thai traditional medicine [25,26].In addition, TLE contains phenolic compounds, which are involved in anti-inflammation and antioxidants [27,28].Several papers have reported that apigenin, one of the flavonoid compounds in TLE [29], has antioxidant [30] and anticancer properties [31].The main toxicity of AFB 1 is oxidative stress occurrence via reactive oxygen species production [32].We hypothesized that TLE had the potential to inhibit aflatoxicosis through its antioxidant ability.In addition, there is no available data about the effects of these herbal medicine products on the duck.Therefore, the poultry industry should develop alternative strategies for detoxifying mycotoxins by TLE in ducks.Hence, the objectives of this research are to comprehensively assess the effects of AFB 1 along with TLE as a natural feed additive in duckling diet on the growth performance, serum biochemical parameters, intestine morphology, carcass traits, meat quality, and immunity responses of Cherry Vally ducks.

Growth Performance
The average daily gain (ADG), the average daily feed intake (ADFI), and the feed conversion ratio (FCR) are presented in Table 2.The final body weight (BW) and ADG were significantly (p < 0.05) reduced by AFB 1 during the growth phase (7 to 42 days).However, feeding T. laurifolia extract and commercial mycotoxin binder along with AFB 1 significantly improved ADG during days 7-42.There was no significant difference in ADFI between the groups.The FCR during 7 to 42 days was significantly (p < 0.05) higher in the AFB 1 -challenged groups.Nevertheless, feeding T. laurifolia extract and a commercial mycotoxin binder significantly enhanced the FCR compared to AFB 1 -fed birds, and it was comparable to that of ducks in the control group.

Blood Biochemistry
Aflatoxin B 1 exhibited significant toxic effects by significantly increasing (p < 0.05) the levels of total cholesterol, triglyceride, aspartate transaminase (AST), and globulin (Table 3) in serum biochemical values.When the AFB 1 -contaminated diet was supplemented with 100 and 200 mg/kg of TLE or 0.5 g/kg of commercial binder, lower concentrations of AST were observed in the serum of the ducklings compared to those fed without these detoxifying agents (p < 0.001).Additionally, the AST values in ducklings fed the AFB 1contaminated diet with TLE treatment were significantly reduced compared to those of the commercial binder treatment.

Intestine Morphology
Aflatoxin B 1 had significantly unequal effects on the different parts of the examined intestine morphology (p < 0.01, Table 4).In general, the villus height (VH) of the duodenum, jejunum, and ileum in ducklings fed the diet containing 0.1 mg/kg AFB 1 was higher than those of ducklings fed the control diet.However, all detoxifying treatments did not decrease the VH but rather increased the values.Notably, AFB 1 increased crypt depth (CD) in the duodenum and jejunum (p < 0.0001) but reduced CD in the ileum (p = 0.0011), while those fed with the AFB 1 -contaminated diet supplemented with 200 mg/kg TLE improved these phenomena.The toxicity of AFB 1 yielded contrasting results in the villus width (VW) of the duodenum and jejunum.Compared to the control group, the VW in the duodenum of the AFB 1 -contaminated group was higher (p = 0.0015), while the VW in the jejunum of the AFB 1 -contaminated group was lower (p = 0.0028).Additionally, 200 mg/kg TLE ameliorated AFB 1 toxicity in VW of both parts of the intestine.As for villus height per crypt depth ratio (VH:CD), only the ileum was affected by AFB 1 (p = 0.013), while the treatments of 100 mg/kg TLE and 0.5 g/kg commercial binder treatments rather increased the ratio.Light microscopy micrographs of the intestine of each experimental group were shown in Figure 1.It was observed that the photomicrograph of the jejunum sections of the control group (T1) showed normal histology of intestinal villi with normal pseudostratified epithelium with goblet cells.In contrast, the addition of 0.1 mg/kg AFB 1 had a significant effect on jejunum tissue histopathology.The photomicrograph of the jejunum section of the T2 group (0.1 mg/kg AFB 1 ) showed mucosal necrosis.Meanwhile, the photomicrograph of the jejunum section of the T3, T4, and T5 groups (AFB 1 with TLE or commercial binder) showed a marked improvement in mucosal necrosis with an increase in villi integrity, especially in T4 (0.1 mg AFB 1 /kg and 200 mg TLE/kg) and T5 (0.1 mg AFB 1 /kg and 0.5 g/kg of commercial binder).There were similar results in the ileum sections.The T1 group had relatively complete and compact villus tissue.The T2 group had a looser villus structure than the T1 group due to aflatoxicosis in the ileum villus structure.The T4 and T5 groups had the effect of improving AFB 1 toxicity.
Light microscopy micrographs of the intestine of each experimental group were shown in Figure 1.It was observed that the photomicrograph of the jejunum sections of the control group (T1) showed normal histology of intestinal villi with normal pseudostratified epithelium with goblet cells.In contrast, the addition of 0.1 mg/kg AFB1 had a significant effect on jejunum tissue histopathology.The photomicrograph of the jejunum section of the T2 group (0.1 mg/kg AFB1) showed mucosal necrosis.Meanwhile, the photomicrograph of the jejunum section of the T3, T4, and T5 groups (AFB1 with TLE or commercial binder) showed a marked improvement in mucosal necrosis with an increase in villi integrity, especially in T4 (0.1 mg AFB1/kg and 200 mg TLE/kg) and T5 (0.1 mg AFB1/kg and 0.5 g/kg of commercial binder).There were similar results in the ileum sections.The T1 group had relatively complete and compact villus tissue.The T2 group had a looser villus structure than the T1 group due to aflatoxicosis in the ileum villus structure.The T4 and T5 groups had the effect of improving AFB1 toxicity.

Carcass Trait, Relative Organ Weight, and Meat Quality
The T. laurifolia extract and AFB1 supplementation did not influence the relative weight of carcass (excluding neck and feet), breast meat, bursa of Fabricius, or spleen, but there was a tendency for an increase (p < 0.1) in liver and gizzard weight.The relative weight of the bursa of Fabricius, spleen, breast meat, and carcass (excluding the neck and feet) was not affected by the T. laurifolia extract or AFB1 supplementation; however, there was a tendency for the liver and gizzard weight to increase (p < 0.1) in duckling fed with T2 and T3 (Table 5).Dietary treatments did not affect the pH test for 45 min and 24 h, thiobarbituric acid reactive substances (TBARS), lightness (L), redness (a), or drip loss (Table 6).However, the inclusion of AFB1 increased (p < 0.05) shear force and breast meat yellowness (b).

Carcass Trait, Relative Organ Weight, and Meat Quality
The T. laurifolia extract and AFB 1 supplementation did not influence the relative weight of carcass (excluding neck and feet), breast meat, bursa of Fabricius, or spleen, but there was a tendency for an increase (p < 0.1) in liver and gizzard weight.The relative weight of the bursa of Fabricius, spleen, breast meat, and carcass (excluding the neck and feet) was not affected by the T. laurifolia extract or AFB 1 supplementation; however, there was a tendency for the liver and gizzard weight to increase (p < 0.1) in duckling fed with T2 and T3 (Table 5).Dietary treatments did not affect the pH test for 45 min and 24 h, thiobarbituric acid reactive substances (TBARS), lightness (L), redness (a), or drip loss (Table 6).However, the inclusion of AFB 1 increased (p < 0.05) shear force and breast meat yellowness (b).

Expression of Immune Response and Metabolizing Cytochrome P450 Enzyme-Related Genes
T. laurifolia extract mitigated liver pathological damage caused by AFB 1 in ducklings.The mRNA levels of the inflammation-related gene (TNFα) in the liver were significantly upregulated in ducks treated with AFB 1 compared to those in the control and TLE groups (Figure 2).Additionally, the mRNA expression levels of CYP1A1 and CYP1A2 in the liver were increased in the AFB 1 group compared with those of the control group.

Antioxidative Capacity of T. laurifolia Extract
The T. laurifolia extract is a traditional Thai herbal medication known for its antioxidative capacity [33].One of the main active ingredients of TLE is total phenolic compounds.A previous study [34] indicated a positive correlation exists for other antioxidant capacity methods, such as DPPH and FRAP with polyphenols.The present examination not only investigated the antioxidative capacity of TLE by determining the ABTS, DPPH, and FRAP but also tested the active compound phenolic content.The TLE of the current study exhibited lower activities in terms of DPPH, ABTS, and total phenolic compounds compared to another study [35].While phytobiotics offer various significant benefits for livestock health, their drawback lies in the variability of composition influenced by factors such as harvesting season and geographical location [36].This variability may also be one of the reasons why a higher concentration (200 mg/kg TLE) was required to have a noticeable AFB1 detoxification effect in this trial.

Aflatoxin B1 Toxicity on Growth Performance
The regulatory limit for AFB1 in the EU, FDA, and China is 0.02 mg/kg for ducklings [37-39].However, this limit level serves as a precautionary measure to prevent the potential harmful accumulation of AFB1 in the bodies of animals after long-term ingestion (over four weeks).Previous research has indicated that AFB1 concentration can impair duck production, and significant hepatic lesions can occur at levels as low as 0.5 mg/kg for a short period (lower than four weeks) [40,41].Taking into account the treatment period (five weeks), experimental efficiency, and various national regulations, we compromised and chose 0.1 mg/kg as the tested content.
It is well established that AFB1 can interfere with poultry energy metabolism, reducing growth efficiency [36,37].Among poultry, meat ducks are susceptible to aflatoxins.A diet containing a high concentration of AFB1 can cause acute death in meat-type ducks, while prolonged exposure to low levels of AFB1 can induce chronic toxicity, resulting in

Antioxidative Capacity of T. laurifolia Extract
The T. laurifolia extract is a traditional Thai herbal medication known for its antioxidative capacity [33].One of the main active ingredients of TLE is total phenolic compounds.A previous study [34] indicated a positive correlation exists for other antioxidant capacity methods, such as DPPH and FRAP with polyphenols.The present examination not only investigated the antioxidative capacity of TLE by determining the ABTS, DPPH, and FRAP but also tested the active compound phenolic content.The TLE of the current study exhibited lower activities in terms of DPPH, ABTS, and total phenolic compounds compared to another study [35].While phytobiotics offer various significant benefits for livestock health, their drawback lies in the variability of composition influenced by factors such as harvesting season and geographical location [36].This variability may also be one of the reasons why a higher concentration (200 mg/kg TLE) was required to have a noticeable AFB 1 detoxification effect in this trial.

Aflatoxin B 1 Toxicity on Growth Performance
The regulatory limit for AFB 1 in the EU, FDA, and China is 0.02 mg/kg for ducklings [37][38][39].However, this limit level serves as a precautionary measure to prevent the potential harmful accumulation of AFB 1 in the bodies of animals after long-term ingestion (over four weeks).Previous research has indicated that AFB 1 concentration can impair duck production, and significant hepatic lesions can occur at levels as low as 0.5 mg/kg for a short period (lower than four weeks) [40,41].Taking into account the treatment period (five weeks), experimental efficiency, and various national regulations, we compromised and chose 0.1 mg/kg as the tested content.
It is well established that AFB 1 can interfere with poultry energy metabolism, reducing growth efficiency [36,37].Among poultry, meat ducks are susceptible to aflatoxins.A diet containing a high concentration of AFB 1 can cause acute death in meat-type ducks, while prolonged exposure to low levels of AFB 1 can induce chronic toxicity, resulting in growth retardation and reduced production [42].Previous research has indicated that poultry-fed diets containing aflatoxins as low as 0.3 mg/kg started to show reductions in growth rate, and feed intake and feed efficiency worsened [43].In the current study, the results indicated that a diet containing 0.1 mg/kg of AFB 1 led to a reduction in ADG and poor FCR in ducklings.Unlike ADG and FCR, the ADFI of ducklings remained unaffected by AFB 1 toxicity, which aligns with the effects of AFB 1 on early young broiler research [44].

Aflatoxin B 1 Toxicity on Serum Biochemical Parameters
Hepatotoxicity is the primary characteristic of AFB 1 toxicity in numerous animal species [5].Blood AST, ALT, and alkaline phosphatase (ALP) levels are commonly used as indicators when measuring the effects of aflatoxin on liver toxicity in poultry [45].Globulin involves several physiological processes, including lipid transportation in birds [15].Our study revealed that AFB 1 altered serum biochemical parameters, leading to significantly higher levels of total cholesterol, triglycerides, AST, and globulin.However, the levels of ALT and ALP in the AFB 1 group did not show a significant increase compared to the control group in our study.This may be attributed to the AFB 1 concentration in this research not reaching the toxic level required for severe liver damage, which would release high amounts of ALT and ALP.The results of the relative liver weight in our experiment support this.Although the liver weights of the AFB 1 group were heavier than those of the control and other treatment groups, the difference was not statistically significant.Similar results were observed in other experiments.For instance, adding over 0.5 mg/kg of AFB 1 to broiler diets can increase serum ALP, ALT, and AST activities [46].However, when the dietary AFB 1 concentration was lower than 0.03 mg/kg, only serum AST levels were significantly increased in broilers [5].The AFB 1 -induced increase in serum total cholesterol and triglycerides observed in this study is consistent with previous research findings [47,48].The liver plays a crucial role in blood fatty acid metabolism [49], while AFB 1 induces liver damage and can lead to abnormal triglyceride metabolism.

Aflatoxin B 1 Toxicity on Intestine Morphology
Aflatoxin B 1 can alter intestinal morphology, leading to reduced nutrient absorption and subsequent growth retardation [50,51].However, the effects of AFB 1 toxicities on poultry intestinal morphology are not entirely clear.This lack of clarity may stem from differences in the specific sections of the intestine, tested variables, and exposure time in previous studies [46].Additionally, the species and age of poultry used in various studies may also play crucial roles in the intestine's response to chronic aflatoxicosis.An earlier study indicated that AFB 1 can induce morphological alterations of the intestinal epithelium by increasing the depth of the crypts, particularly in the small intestine (duodenum and jejunum) [52].While these findings were consistent with the observations in the duodenum and jejunum, they did not align with those of the ileum in the present study.Furthermore, most research has indicated that AFB 1 decreased VH in the small intestine of broilers.However, contrary to the observations in broilers [46,52], AFB 1 had no effect on VH in laying hens [53].The results of our meat duckling trial also differed from those of the broiler chicken test.Surprisingly, the VH of the duodenum, jejunum, and ileum were all significantly increased by AFB 1 toxicity.Alterations in both the height and width of villi were also noted in ducks treated with AFB 1 .The alterations in the structure of villi were a result of the activation of the apoptotic pathway by AFB 1 , which subsequently may be related to the absorption of nutrients.Given the differences in these results of intestinal morphology, in addition to the abovementioned differences in varieties and sampling locations, further testing may be necessary to verify and confirm these findings.

Aflatoxin B 1 Toxicity on Carcass Traits and Meat Quality
Several interesting results were observed regarding carcass traits and meat quality.In contrast to other reports [5,10], our results did not show significant changes in the relative weights of the liver and other organs.Although there was a slight increase in the AFB 1 -contaminated treatment group compared with the control group, this difference did not reach statistical significance.This could be attributed to the tested concentrations of AFB 1 in this study causing mild hepatotoxicity that did not reach the threshold to alter liver weight.In terms of meat quality, it was discovered that the color of the meat in the AFB 1 -contaminated group showed a significant increase.To the best of our knowledge, there were no other poultry reports that investigated whether AFB 1 changes the color of poultry meat.However, we found a sheep report [54] indicating that AFB 1 altered the lightness (L value) of the meat but not the yellowness (b value).Although there were slight differences between the results of the former study and ours, these variations may be attributed to differences in animal species.Nonetheless, it is plausible that AFB 1 could indeed cause changes in meat color.We speculated that disruptions in pigment metabolism and inflammatory responses associated with liver damage could also influence the color of the meat, potentially contributing to changes in its yellowness [55].

Aflatoxin B 1 Toxicity on Immunity and Cytochrome P450 Enzyme-Related Genes
Aflatoxin B 1 induces oxidative damage and apoptosis in hepatocyte cells and is primarily metabolized by cytochrome P450 (CYP450) enzymes [56].In poultry liver, AFB1 is bioactivated by enzymes such as CYP1A1, CYP1A2, and other enzymes (e.g., CYP2A6 and CYP3A4).CYP450 enzymes convert AFB 1 into an electrophilic, highly reactive, and unstable metabolite known as aflatoxin-8,9-epoxide (AFBO) [57,58].This metabolite can interact with cellular macromolecules, binding to guanine residues in DNA, causing genotoxicity, and reacting with proteins to induce cytotoxicity [59].These interactions result in irreversible DNA damage and can lead to hepatocarcinoma in humans, primates, and ducks [60].Consistent with previous research in broiler chickens [61], our study observed that AFB 1 exposure led to a significant increase in CYP1A1 and CYP1A2 mRNA expression.Additionally, our findings were consistent with previous studies, which demonstrated that AFB 1 treatment increased the mRNA levels of TNF-α [62,63].This indicates that AFB 1 toxicity induces the immune response and inflammatory cytokine production in ducklings.However, the mRNA expressions of these enzymes and TNF-α were lower in groups treated with TLE and a commercial binder, suggesting that these feed additives effectively neutralize the hepatotoxic effects of AFB 1 .

Antimycotoxigenic Efficacies of Thunbergia laurifolia Lindl.
Aflatoxin B 1 is primarily metabolized through CYP1A1 and CYP1A2, producing a highly reactive intermediate (AFBO), which induces the formation of reactive oxygen species (ROS) within hepatocytes [64].The accumulation of ROS leads to oxidative stress, characterized by an imbalanced response between the production of reactive species and the ability of cells to detoxify or repair the damage [65].Reactive oxygen species damage cellular components, including lipids, proteins, and DNA, initiating lipid peroxidation and compromising membrane integrity, ultimately leading to cell damage and death [66].Oxidative damage and cellular stress induce a series of inflammatory responses in the liver, further aggravating tissue damage.Liver damage impairs critical functions, such as detoxification, protein synthesis, and nutrient metabolism, leading to reduced nutrient absorption and utilization, which contributes to poor growth performance [67].
Therefore, T. laurifolia with natural antioxidants may be a promising option to neutralize AFB 1 toxicity.Much research has indicated that T. laurifolia possesses antioxidant and anti-inflammatory properties, as well as anticancer activities, due to its ability to increase catalase (CAT) and glutathione peroxidase (GPx) activities, thereby removing ROS [68][69][70].Previous research on chickens has shown promising results using 2% T. laurifolia leaf [71].This treatment ameliorated the adverse effects of multiple mycotoxin-contaminated feeds, improving nutrient digestibility and increasing the activity of glutathione peroxidase.However, it did not lead to a significant change in the growth rate.Our research further investigated the potential of TLE in mitigating the effects of AFB 1 on growth reduction and hepatoxicity.By utilizing extracts of T. laurifolia in our study, we hypothesized that some impurities were eliminated to enhance the concentration of bioactive chemicals, such as total phenolic compounds [72].Therefore, we only used 100 mg/kg TLE to improve the growth reduction caused by AFB 1 , and the treatment of 200 mg/kg TLE had a stronger detoxification ability, as observed in growth performance, serum biochemical traits, intestinal morphology, and meat quality.
Our results suggest that supplementing TLE into duckling diets could be a natural and effective detoxifying agent against AFB 1 contamination.This can lead to improved growth performance, feed efficiency, and overall health in poultry, which is crucial for the poultry industry.Additionally, the study presented that TLE improves meat quality by mitigating the adverse effects of AFB 1 .This is critical for ensuring that the meat produced is safe and high quality.Our findings pave the way for further research into the use of TLE for detoxifying various mycotoxins in different animal species.

Conclusions
It can be concluded that dietary supplementation of T. laurifolia extract in ducklings ameliorated the adverse effects of AFB 1 on growth performance, alleviated liver damage by increasing the drug-metabolizing enzymes (Cytochrome P450), and improved the intestinal health of ducks through participation in their detoxification.

Animal and Ethical Approval
A total of 180 seven-day-old Cherry Valley ducks were obtained from the Faculty of Agriculture, Chiang Mai University, Thailand.The ducks were housed in pens with strict biosecurity measures, with each treatment containing 3 replications of 12 birds each.Over the 35-day duration of the experiment, the ducks received water and feed ad libitum (Table 7).All experimental procedures in this study were conducted strictly in accordance with the recommended guidelines and were submitted for ethical approval by the Animal Ethics Committee, Faculty of Agriculture, Chiang Mai University.

Plant Materials
The mature leaves of T. laurifolia Lindl.were collected from Hangdong District, Chiang Mai Province, Thailand.The leaves were cleaned, chopped into pieces, and then oven dried at 60 • C for 24 to 48 h.Subsequently, the dried leaves were powdered using a dry grinder to obtain particles of approximately 0.2 mm in size.The powdered material was stored in a light-resistant container until it was used for the extraction studies.

Extraction Method and Phenolic Content Measurement
The procedure involved soaking the powdered T. laurifolia leaves in boiling distilled water (1:10 w/v) for one hour.Subsequently, the mixture was passed through a filter paper (Whatman No. 41) and three layers of gauze.The filtrate obtained was freeze-dried and kept in a desiccator at a temperature of 4 • C. To facilitate future use, the extract was diluted in distilled water to achieve the appropriate concentrations and then stored at a temperature of −20 • C. The Folin-Ciocalteu technique [73] was employed to quantify the total phenolic content.The extract was combined with the Folin-Ciocalteu reagent and a 7.5% (w/v) solution of NaCO 3 .The calibration standard for gallic acid was established by incubating it for 60 min and using a UV-Vis spectrophotometer (SPECTROstar Nano, BMG LABTECH, Ortenberg, Germany).The extract's total phenolic content was determined in milligrams of gallic acid per gram.

Antioxidative Assays
The DPPH and ABTS radical scavenging activities were evaluated using modified methods based on Sunanta et al. [74] and Sangta et al. [44], respectively.For the DPPH assay, 25 µL of the extract was mixed with 250 µL of 0.20 mM DPPH (2,2-diphenyl-1picrylhydrazyl) solution.The mixture was then incubated at room temperature, in the dark, for 30 min, and the absorbance was measured at 517 nm.Regarding the ABTS assay, 200 µL of the extract was mixed with 500 µL of a working solution containing 7.00 mM ABTS [2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)] and 2.45 mM potassium persulfate.The mixture was incubated in the dark at room temperature for 12-16 h, and the absorbance of the samples was measured at 734 nm.The FRAP was determined using the modified Aljadai method [75].In this method, 10 µL of the extract was mixed with 190 µL of FRAP reagent for 30 min in the dark, and the absorbance was measured at 593 nm using ascorbic acid as a standard reference.

Treatment Diet Preparation
The powder of AFB 1 standard (purity ≥ 98%) and commercial binder (Mycosorb Advance) were purchased from Sigma (Saint Louis, MO, USA) and American Colloid Company (Lovell, WY, USA), respectively.One milligram of AFB 1 standard was dissolved in 100 mL of 95% ethanol (Merck, Darmstadt, Germany) to obtain 10 mg/kg AFB 1 stock solutions.The prepared solution was then sprayed evenly on the basal feed and mixed to obtain the 0.1 mg/kg AFB 1 -contaminated diet [76,77].The equivalent amount of ethanol without AFB 1 solution was sprayed evenly on the basal feed to obtain the control diet.The treatment concentration of TLE and the commercial binder were calculated, respectively, added uniformly to the diet, and mixed evenly.Mycotoxins were detected in the basal diet using ELISA kits (R-Biopharm, Darmstadt, Germany).The analysis revealed that the quantities present in the sample were as follows: 0.012 mg/kg AFB 1 , 0.0212 mg/kg T-2 toxin, 0.015 mg/kg ochratoxin A, 0.035 mg/kg zearalenone, and 0.015 mg/kg deoxynivalenol, respectively.

Growth Performance
All ducklings were fed treatment diets for 35 days.The ducks were clinically observed at least twice daily, and mortality was recorded.Furthermore, the ducks were individually weighed on the age of day 7 and day 42.The performance variables measured in this study include BW, ADG, ADFI, and FCR.

Characteristics
Blood samples were collected at day 42 from each treatment (6 birds) for biochemical analyses.The blood samples were then centrifuged at 3000× g for 15 min, and the serum was separated to determine liver function parameters such as AST, ALT, ALP, total protein, globulin, and albumin.All blood characteristics were measured using a BioMajesty ® JCA-BM6010/C kit from DiaSys Diagnostic Systems (Holzheim, Germany) with an automated chemistry analyzer BX-301 (Asia Green, Singapore).

Relative Organ Weight
Following the bleeding process, all ducks from each treatment were euthanized via cervical dislocation.Subsequently, the liver, kidney, heart, spleen, gizzard, and bursa of Fabricius were removed, and their weights were measured.The organs were weighed, and their weights were represented as relative organ weights: Relative weight = (Organ weight)/(Final BW) × 100.

Carcass and Meat Quality
After 42 days of testing, each duck was carefully weighed before being exsanguinated and sacrificed via cervical dislocation.The weight of the carcass (excluding the neck and feet), breast meat, liver, gizzard, pancreas, thymus, bursa of Fabricius, spleen, and abdominal fat was extracted and measured after being rinsed with saline solution.Organ size was quantified as a proportion of BW.The pH of the breast meat was determined using a calibrated glass-electrode pH meter (WTW pH 340-A, WTH Measurement Systems Inc., Ft. Myers, FL).The lightness (L*), redness (a*), and yellowness (b*) values of the breast meat were measured using a Minolta CR410 Chromameter from Konica Minolta Sensing Inc., located in Osaka, Japan.The water-holding capacity (WHC) was determined following the procedures outlined by Kauffman et al. [78].Additionally, the drip loss was quantified using roughly 2 g of heated material, following the plastic bag technique outlined by Honikel [79].Subsequently, the cooking loss was calculated using the methodology laid out by Sullivan et al. [80].The TBARS were quantified using the technique outlined by Witte et al. [81], with the results expressed as milligrams of MDA per kilogram of muscle.The extraction process involved the use of a solution of trichloroacetic acid with a concentration of 20% by weight/volume.

Immune Response and Metabolizing Cytochrome P450 Enzyme-Related Genes Expression in the Liver
At the end of the experiment, three birds were randomly selected from each treatment, and their liver tissues were immediately removed and frozen at −80 • C until RNA extraction.Total RNA was extracted from 50 mg of liver samples homogenized with liquid nitrogen using Trizol and a columnar RNA extraction kit (Invitrogen, PureLink TM RNA Mini Kit, Thermo Scientific, Wilmington, NC, USA) according to the manufacturer's protocol.The extracted RNA was quantified using a spectrophotometer (NanoDropTM 2000, Thermo Scientific, Wilmington, NC, USA) at an absorbance ratio of 260-280 nm.Subsequently, the cDNA was synthesized using a cDNA synthesis kit (iScriptTM cDNA Synthesis Kit, BIO-RAD, Hercules, CA, USA) according to the manufacturer's instructions.
The qPCR reaction was carried out using the CFX ConnectTM Real-Time PCR System (BIO-RAD, Hercules, CA, USA) with the iTaq Universal SYBR Green supermix 2X (BIO-RAD, Hercules, CA, USA) and specific primers for individual genes (Table 8).Changes in the expression levels of the above genes were measured using the 2-∆∆Ct method and a standard curve, as outlined by Larionov et al. [82].

Statistical Analysis
The experimental data were analyzed using the analysis of variance (ANOVA) procedure of SAS Enterprise Guide Software V.9.4 (SAS Institute, Cary, NC, USA).The least square means (LSM) were compared using Tukey's test, and a probability level of p < 0.05 was considered statistically significant.

Figure 1 .
Figure 1.Histological representations of the H&E-stained jejunum and ileum sections of ducks.(a) T1: Control, only basal diet without AFB1, TLE, or commercial binder, which showed normal histology of intestinal villi with normal pseudostratified epithelium with goblet cells (arrow) in jejunum; (b) T2: the basal diet containing 0.1 mg AFB1/kg, which AFB1 showed significant mucosal necrosis and decreased villi integrity in the jejunum (arrow); (c) T3: the basal diet containing 0.1 mg AFB1/kg and 100 mg TLE/kg, which showed mild mucosal necrosis and loose villi integrity in the jejunum (arrow); (d) T4: the basal diet containing 0.1 mg AFB1/kg and 200 mg TLE/kg, which showed slight mucosal necrosis and loose villi integrity in the jejunum (arrow); (e) T5: the basal diet containing 0.1 mg AFB1/kg and 0.5 g/kg of commercial binder, which showed slight mucosal necrosis and loose villi integrity in the jejunum (arrow).(f) T1 showed the complete and compact villus tissue in the ileum (arrow); (g) T2 showed loose villus structure in the ileum (arrow); (h) T3 showed slightly loose villus structure in the ileum (arrow); (i) T4 showed slightly loose villus structure in the ileum (arrow); (j) T5 showed slightly loose villus structure in the ileum (arrow); Magnification was 10× the objective lens.Scale bars represent 100 μm.

Figure 1 .
Figure 1.Histological representations of the H&E-stained jejunum and ileum sections of ducks.(a) T1: Control, only basal diet without AFB 1 , TLE, or commercial binder, which showed normal histology of intestinal villi with normal pseudostratified epithelium with goblet cells (arrow) in jejunum; (b) T2: the basal diet containing 0.1 mg AFB 1 /kg, which AFB 1 showed significant mucosal necrosis and decreased villi integrity in the jejunum (arrow); (c) T3: the basal diet containing 0.1 mg AFB 1 /kg and 100 mg TLE/kg, which showed mild mucosal necrosis and loose villi integrity in the jejunum (arrow); (d) T4: the basal diet containing 0.1 mg AFB 1 /kg and 200 mg TLE/kg, which showed slight mucosal necrosis and loose villi integrity in the jejunum (arrow); (e) T5: the basal diet containing 0.1 mg AFB 1 /kg and 0.5 g/kg of commercial binder, which showed slight mucosal necrosis and loose villi integrity in the jejunum (arrow).(f) T1 showed the complete and compact villus tissue in the ileum (arrow); (g) T2 showed loose villus structure in the ileum (arrow); (h) T3 showed slightly loose villus structure in the ileum (arrow); (i) T4 showed slightly loose villus structure in the ileum (arrow); (j) T5 showed slightly loose villus structure in the ileum (arrow); Magnification was 10× the objective lens.Scale bars represent 100 µm.

Table 1 .
Total phenolic compounds and antioxidant activity of T. laurifolia extract.

Table 2 .
Effects of T. laurifolia extract on growth parameters of aflatoxin B 1 -challenged ducklings.

Table 3 .
Effects of T. laurifolia extract on serum biochemical of aflatoxin B 1 -challenged ducklings.

Table 4 .
Effects of T. laurifolia extract supplementation on intestinal morphology of aflatoxin B 1challenged ducklings.

Table 5 .
Effects of T. laurifolia extract on carcass trait and relative organ weight of aflatoxin B 1challenged ducklings.

Table 6 .
Effect of T. laurifolia extract on meat quality of aflatoxin B 1 -challenged ducklings.

Table 7 .
The formulation and proximate composition of the experimental diet (g/kg).

Table 8 .
Primer sequences, amplicons, and the related information for quantitative real-time PCR.